We propose to develop a biochemical approach for probing the involvement of phospholipids in the functioning of biological membranes. The method is based on the replacement of the polar headgroup of the phospholipids of E. coli by their phosphorothioate analogs. This will be achieved by culturing strains of E. coli, which are constitutive for glycerolphosphate (GOP) transport, in the presence of sn-glycerol 3-phosphorothioate (GSP). The rationale behind this approach is that the phosphorothioate analog will be transported into E. coli, incorporated into phospholipids and that the phospholipid analogs will effectively mimic the action of the parent compounds in the membrane. The replacement of a non-bridge oxygen atom by a sulfur atom can be considered a minimal structural alteration to the parent oxygen compound. However, since phosphorothioates are, in general, less susceptible to hydrolytic cleavage by phosphatases and by phosphodiesterases than their "all oxy" counterparts, blocks may occur in certain steps in phospholipid metabolism, resulting in an abnormal membrane state and, in consequence, lead to alterations in specific cellular processes. The overall objectives of this proposal are to study the metabolism of sn-glycerol 3-phosphorothioate in E. coli and to determine its biochemical effects on membrane and cell functions.